High - Level Expression Of Phytase In Corydalis Virginiana In Pichia Pastoris

Phytase, a feed additive, can improve the utility of phosphate in plant feedstuffs and the absorption of nutrients for mono-gastric animals. It can also reduce the phosphorus pollution in environment. Phytase has a bright and broad application prospects because that feeding effects had been acknowleged. However, there are two major drawbacks to affect the wide use of phytase:one is the phytase lower production for wild-type strain; another is the thermostability that affect the commercial use. Therefore, increasing the yield and improving the thermostability phytase need further research.The 6-phyA gene was artificially synthesized based on codon preference of Pichia pastoris using the technique of overlapping PCR. The synthesized 6-phyA was inserted into the vector of pPIC9K to construct the 6-phyA integrative expressing vector. This vector was transformed into the host Pichia pastoris of GS115 by electroporation and the high copy of Mut+transformants of 6-phyA were selected. We have developed a strain of Pichia Pastoris having ability of over expressing peniophora lycii phytase. By this study, the following results were achieved:1.According to the GenBank sequence of peniophora lycii phytase, fourty four primers were used to have synthesized 6-PhyA gene successfully using the overlap extension PCR technology.The result of analysis for codon preference of Pichia pastoris is ideal, and the coding-sequence homology was 100%.2.Through the selection of signal peptide research, we attempt to fuse 6-PhyA itself signal peptide together with a-signal peptide to promote the expression of phytase.however the effect is not ideal. By researching the 6-PhyA wild type signal peptide in saccharomyces cerevisiae, we founded that 6-PhyA can not be expressed in this yeast cells under the strong promoter of GDP driver. This proved that the wild type signal peptide cannot make the phytase to secreting expression.3. The Pichia painstoris expression vector, pPIC9K, was successfully co nstructed. Then the high copy transformant named GS115/pPIC9K/L6ph was scr eened out after the plasmid was eletro-transformed into Pichia pastoris, GS115. After being induced by methanol for 72h, the phytase activity in fermented br oth was approximately 23.7U/mL.It indicated that the PhyA gene was overexpr essed in Pichia pastoris.4. Assay of the enzymological characteristics showed that the analysis of SDS-PAGE found d that its molecular weight was approximately 50kD. The optimal temperature for the phytase was 37℃ and the phytase displayed maximumactivity at pH 5.5, which were suitable for single stomach animal digestive tract environment. After heat treatment for 60 minutes at 55℃,6-PhyA maintained 44% of their initial activities.More than 55 ℃ heat treatment, enzyme activity were declined sharply. However, the metal ion has no affects on the activity of phytase.